Developing non-cognitive approaches for treating obesity is imperative. We have been using gnotobiotic mice to examine the significance of human-bacterial symbioses in the gut, and discovered that the intestinal microbiota has a remarkable effect on fat storage. Colonization ('conventionalization') of adult germ-free (GF) C57B1/6 (B6) mice with a cecal microbiota harvested from conventionally-raised mice produces a 60% increases in total body fat content and white adipose tissue (WAT) weight. This rapid increase occurs despite decreased chow consumption and increased metabolic rate, is sustained, and accompanied by increased leptin levels, insulin resistance, and increased hepatic lipogenesis. The lipogenic response is associated with increased nuclear import of carbohydrate response element binding protein (ChREBP), modest increases in insulin-responsive SREBP-1, and trans-activation of ChREBP/SREBPl lipogenic gene targets. WAT hypertrophy is associated with increased LPL activity and intestine-specific transcriptional suppression of Fiaf (encodes a secreted LPL inhibitor). Moreover, GF Fiaf knockout mice have higher WAT LPL activity and the same body fat content as 'conventionalized'(Fiaf-suppressed) wild-type (wt) littermates: their fat stores are not increased further with conventionalization. These results suggest the following testable hypothesis: (a) microbial processing of otherwise indigestible dietary polysaccharides (PS) provides monosaccharides that lead to ChREBP, and possibly, SREBP-1- stimulation of hepatic lipogenesis: (b) microbial suppression of intestinal Fiaf, combined with the lipogenic response, promotes LPL-mediated increases in adipocyte fat storage;(c) increasing Fiaf expression and/or activity should promote leanness. Aim 1- Use wt B6 mice to determine the role of dietary carbohydrates and microbial ecology on microbiota-induced fat storage. GF and conventionalized mice will be fed isocaloric high PS/low fat, .high fat/low PS, or high sugar/low fat diets, and the effects on body fat content, VO2, feptin/glucose/insulin, WAT LPL, and intestine/liver/muscle energy metabolism assayed. Microbial requirements will be assessed by colonization with all or some components of a simplified 8-member microbiota (Altered Schaedler Flora). Aim 2- Determine the contribution of the microbiota-associated hepatic lipogenic response by conventionalizing GF mice with a ChREBP knockout, a SREBP-1 c knockout, combined ChREBP and SREBP-1 c deficiencies, or a hepatocyte-specific Fasl knockout. Aim 3- Introduce a transgene constitutively expressed in the small intestinal epithelium into Fiaf-/- mice. We predict that these mice will phenocopy leaner GF wt mice, whether or not they have been colonized [proof-of-concept genetic test of whether therapeutic manipulations that increase Fiaf expression (or activity) will promote leanness].